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The Centrosomal Protein CEP350 regulates MT dynamics during epithelial polarization

AutorRíos, Rosa M. ; Gavilán, María P.
Fecha de publicación12-nov-2012
CitaciónIII Workshop of cell adhesion, migration and invasion (2012)
ResumenCEP350/CAP350 is a large non-coiled coil centrosomal protein which contains three Ser-rich regions and a CAP-Gly motif, a specialized protein module able to interact with tubulin and/or microtubules (1). Overexpression of CAP350 produced a massive stabilization of cytoplasmic MTs but not via its CAP-Gly domain. Instead, this activity is localized at its basic Nterminus. Alpha-catenin is a component of the adherens junctions (AJ), cell-cell contacts present in all epithelial cells. It has been recently shown that alpha-catenin is able to promote MT nucleation and/or stabilization in a centrosome-independent fashion (2). A two-hybrid screening for CAP350 has revealed a putative interaction between these two proteins. We hypothesize that MT-stabilization activity of alpha-catenin is mediated by CAP350. Co-immunoprecipitation experiments in several cell types confirmed the CAP350/alpha-catenin interaction in vivo. A systematic truncation analysis revealed that CAP350 contains two alphacatenin binding sites, located at central and C-terminal regions, both of which specifically bind to the VH1 domain of alpha-catenin. By IF, CAP350 was detected not only at the centrosome but also at tips of MT bundles that concentrate at cell-cell contacts in confluent cultures of epithelial cells where alpha-catenin is also present. This localization was dependent on cell-cell contacts and MTs, but not on actin. To test whether alpha-catenin binding to MTs is mediated by CAP350, we carried out MT pelleting assays. In the presence but not in the absence of CAP350, alpha-catenin co-sedimented with MTs in vitro. These results are compatible with a model in which CAP350 targets alpha-catenin to MTs. In CAP350-depleted cells, MTs were not longer attached to the centrosome and they bent underneath the plasma membrane further supporting a role of CAP350 at the cell periphery. Interestingly, CAP350-depleted cells appeared bigger than control cells, and cell-cell contacts were disorganized. These phenotypes are reminiscent to those described in Caco2 cells lacking any of the proteins involved in the anchoring of MTs to adherens junctions (3). Finally, to evaluate the role of CAP350 in cell polarity, we analysed the effect of CAP350 depletion in the MDCKII cell system. In 2D MDCKII cultures, CAP350-depleted cells failed to properly polarized: cell-cell contacts appeared wide and undefined, the apical-basal array of MTs was completely absent, and the cells did not adopt a columnar shape but rather they were planar and much bigger. MDCKII cysts formed from CAP350-depleted cells contained multiple small lumens or even no lumen. Altogether, the results obtained form MDCKII studies reveal a role of CAP350 in epithelial morphogenesis probably by regulating MT dynamics.
DescripciónPóster presentado en el III Workshop of cell adhesion, migration and invasion, celebrado en Begur (Girona) del 12 al 14 de noviembre de 2012
URIhttp://hdl.handle.net/10261/123909
Aparece en las colecciones: (CABIMER) Comunicaciones congresos
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